Theory and experiment will be combined to study three unanswered questions regarding the bacterial chemotaxis signaling pathway: (i) the mechanism of signal amplification and integration in receptor clusters localized at cell poles, (ii) the mechanism of motor switching, which results from interactions between the response regulator protein CheY and the switch complex of the flagellar motor, and (iii) how these and other mechanisms contribute to, and are affected by signal fluctuations in the pathway. The project is designed to incorporate the answers to each of these questions, as they are resolved, into the most detailed computational model of the bacterial chemotaxis pathway to date. A specific focus will be on extending the fluorescence resonance energy transfer (FRET) system developed by Berg and colleagues for real-time measurements of the intracellular signaling state. The aim is to enable such measurements in single cells over extended periods, which would allow for direct comparisons with stochastic simulations of the pathway. It is expected that the methods developed will be applicable to many other cell signaling systems, and the results obtained should provide insight into the nature of other mesoscopic phenomena in the cell.